Amplification of minute amounts of nucleic acid sequences by techniques such as the polymerase chain reactions is an established method, widely used for example in routine diagnostics and research laboratories. A standard nucleic acid amplification reaction consists of repeatedly copying a target sequence and the produced copies generating an exponential multiplication process of the target sequence. After several tens of multiplication cycles the nucleic acid sequences can be detected and their amount measured by using a label that attaches to the copied sequence and detecting that label. Commonly used labels are fluorescent molecules, radioisotopes and chemiluminescent molecules.
The most common nucleic acid amplification reaction is the polymerase chain reaction (PCR). PCR consists of repeatedly cycling the reaction volume through different temperatures to 1) denaturate the formed nucleic acid double stranded helixes, 2) anneal short nucleic acid sequences called primers to the now single stranded halves of the double helix at the ends of the portion of the nucleic acid sequence to be amplified, 3) elongate the still partially single stranded nucleic acid sequences with the help of a special enzyme such as the Taq DNA polymerase, 4) return back to denaturation step of the cycle.
The method according to this invention refers to those fluorometric nucleic acid amplification assays where one of the reacting nucleic acid sequences is attached to microparticles serving as a solid matrix. The solid matrix attached sequence combines with its counterpart from the amplification reaction solution. Detection of the amount of the solid matrix bound nucleic acid sequence follows by detection of increased or decreased fluorescence.
A special problem of the nucleic acid amplification reactions is related to their extreme sensitivity. Any carry-over or any false matching sequence is amplified along with the desired target sequence. These non-target sequences are introduced either as impurities or by miscombination during annealing phases. To avoid such problems the amplification factor should therefore be kept as low as possible and the reaction volume closed throughout the amplification cycles.
Some applications require the quantification of the target. The procedure includes observing the number of cycles as well as the amplification rate from cycle to cycle. Quantitative determination of the target can only be performed during the exponential growth rate of the target product, i.e. when the number of cycles is as low as possible. This type of assay can only be performed effectively if the measurement takes place directly from the reaction volume, i.e. the assay is homogeneous. Most common techniques using fluorescence, radioisotopes or chemiluminescence require stopping of the reaction and performing incubation and washing steps for the assay. Such assays are not optimally suited for quantification purposes since all the steps must be predetermined - precision and dynamics of the quantification is compromised. A known homogeneous assay type suitable for direct measurement of amplification process bases on the use of fluorescence resonant energy transfer (FRET). In this method as a nucleic acid double helix is formed, two labels being parts of the sequence are brought into close proximity. The first, normally fluorescent label is quenched by the second label. The quenching only takes place in close proximity and thus free labels are not affected. The amount of formed amplification product is measured from the drop in total fluorescence signal. An inherent problem in this technique is it's yet low sensitivity thus necessitating a relatively high number of amplification cycles to be performed.
In PCR the sample is cycled through different temperature steps. The speed of reaching each of the temperature levels determines the speed at which the amplification reaction advances. This in turn is directly dependent on the volume of the sample; smaller samples are preferable from this kinetic point of view. Contrary to this kinetic requirement is the detection sensitivity where conventional detection technologies benefit from larger volumes.
There is a constant need for simpler, faster, more cost-effective and more sensitive nucleic acid sequence amplification analyses. The new nucleic acid sequence amplification assay method of the present invention using fluorescent labels enables the direct and sensitive measurement of the amplification reaction without separation and washing steps enabling the on-line monitoring of amplification process in a closed cuvette. The new method has no practical volume limits and is thus well suited for very small volumes. The new method is particularly well suited for using together with the PCR amplification. Further the new method is suited for multiparametric determination of amplification products. Different sequences and control sequences may be monitored from the same amplification volume.
Two-Photon Excitation
Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. According to the concept of probability, the absorption of a single photon in a dye, is an independent event, and the absorption of several photons is a series of single, independent events. The probability of absorption of a single photon can be described as a linear function as long as the energy states that are to be excited are not saturated. The absorption of two photons is a non-linear process. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is a thus a quadratic process.
The properties of the optical system used for fluorescence excitation can be described with the response of the system to a point-like light source. A point-like light source forms, due to diffraction, an intensity distribution in the focal plane characteristic to the optical system (point spread function). When normalized, this point spread function is the probability distribution of how the photons from the light source reach the focal area. In two-photon excitation, the probability distribution of excitation equals the normalized product of intensity distributions of the two photons. The probability distribution thus derived, is 3- dimensional, especially in the vertical direction, and is clearly more restricted than for a single photon. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted 3-dimensional vicinity of the focal point is excited.
When a dye is two-photon excited the scattering of light in the vicinity of the focal point and from the optical components, is reduced remarkably compared to normal excitation. Furthermore, two-photon excitation decreases the background fluorescence outside the focal point, in the surroundings of the sample and in the optics. Since the exciting light beam must be focused onto as a small point as possible, two-photon excitation is most suitable for the observation of small sample volumes and structures, which is also the case in the method according to this invention.
The advantage of two-photon excitation is also based on the fact that visible or near-infrared (NTR) light can, for example, be used for excitation in the ultraviolet or blue region. Similarly, excitation in the visible region can be achieved by NTR light. Because the wavelength of the light source is considerably longer than the emission wavelength of the dye, the scattering at a wavelength of the light source and the possible autofluorescence can be effectively attenuated by using low-pass filters (attenuation of at least 10 orders of magnitude) to prevent them from reaching the detector.
Since in two-photon excitation the density of photons per unit volume and per unit time must be very high in order to make two photons to be absorbed into the same chromophore, it is useful to use lasers which generate short pulses with high repetition rate. A practical laser for two-photon excitation is for example a passively Q-switched Nd:YAG laser with a pulse length of 1 ns.